Summary Reproductive and developmental disorders caused by drugs or environmental chemicals are a prominent health issue worldwide. Current toxicity testing methods set by regulatory agencies predominately rely on animal testing. The cost and time associated with animal testing for risk assessment seriously limit efforts for extensive toxicity screening. These data gaps necessitate the implementation of a new vision for toxicity testing as reflected in the National Research Council?s report ?Toxicity Testing in the 21st Century? and the restructuring of risk assessment procedures. Toxicity testing in the 21st century must move beyond classical methods of risk assessment by incorporating modern molecular knowledge, such as adverse outcome pathways (AOPs) with advanced in vitro models. Currently, in vitro reproductive toxicity testing models are actively being developed. Under the support of R43 ES027374 grant, ReproTOX established a Mini-Testis model from testicular cell lines and found this Mini-Testis model could identify reproductive toxicants. Both morphological and cell-specific biomarkers assessment demonstrated that this co-culture model created an in vitro-like niche, formed a three-dimensional cytoskeleton bundle structure and supported germ cells within the 3D environment. We initially validated this model by discriminating reproductive toxicants among 32 compounds and observed a strong correlation between the in vitro IC50 and in vitro rLOAEL (the lowest observed adverse effect level) for these selected compounds. The calculation of concordance, sensitivity, and specificity further supported the reliability of this model. Our results from the phase I study suggested that our in vitro Mini- Testis model might be a valuable screening tool for reproductive toxicant assessment and to prioritize chemicals for further testing. In this phase II proposal, we will further examine and optimize the protocol for the in vitro Mini-Testis model. We will test the Mini-Testis model with an expanding list of ?reference testing compounds?, and validate blindly our predictive model of pathway-based high-content and high-throughput platform. The Specific Aims are (1) to complete the development of high-content assays based on the adverse outcome pathways associated with the reproductive toxicity in an in vitro Mini-testis model; (2) to apply the multi-parametric high-throughput and high-content analysis (HT/HCA) to examine the sensitivity and specificity through comparison between the IC50 obtained from the in vitro model and in vivo reproductive lowest observed adverse effect level (rLOAEL) to validate the in vitro Mini-testis model; (3) to conduct the intra- and inter-laboratory reproducibility study to validate the in vitro Mini-testis model; we will test the variability within-test and the reproducibility of the test within and among laboratories. Altogether, this proposed Phase II will focus on the continued development of this 3D Mini-Testis screening platform toward a validated quantitative pathway-based HT/HCA screen for reproductive toxicity. Commercialization of this in vitro platform and its ability to screen up to a thousand compounds via focusing on multiple endpoints associated with adverse reproductive effects caused by agents from a variety of chemical classes. This Mini- Testis model-based platform has the potential to offer predictive reproductive and developmental toxicity without the use of animals in a high-throughput format, and will be a critical part of an integrated testing strategy for Reproductive and developmental toxicity.